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. 2017 Apr;20(4):540-549.
doi: 10.1038/nn.4503. Epub 2017 Feb 13.

Cadherins Mediate Cocaine-Induced Synaptic Plasticity and Behavioral Conditioning

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Free PMC article

Cadherins Mediate Cocaine-Induced Synaptic Plasticity and Behavioral Conditioning

Fergil Mills et al. Nat Neurosci. .
Free PMC article

Abstract

Drugs of abuse alter synaptic connections in the reward circuitry of the brain, which leads to long-lasting behavioral changes that underlie addiction. Here we show that cadherin adhesion molecules play a critical role in mediating synaptic plasticity and behavioral changes driven by cocaine. We demonstrate that cadherin is essential for long-term potentiation in the ventral tegmental area and is recruited to the synaptic membranes of excitatory synapses onto dopaminergic neurons following cocaine-mediated behavioral conditioning. Furthermore, we show that stabilization of cadherin at the membrane of these synapses blocks cocaine-induced synaptic plasticity, leading to a reduction in conditioned place preference induced by cocaine. Our findings identify cadherins and associated molecules as targets of interest for understanding pathological plasticity associated with addiction.

Conflict of interest statement

Competing Financial Interests Statement: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Cadherins are expressed in dopaminergic neurons and are essential for LTP in the VTA
(a) VTA neurons co-immunostained for cadherins (green), dopamine transporter (DAT; magenta) and DAPI (blue). Arrowheads indicate neurons positive for both cadherin and DAT, asterisks indicate neurons which are positive for cadherin but not DAT. Scale bar = 10 μm. (b) Spike-timing-dependent LTP in the VTA was abolished by treatment with a peptide containing an HAV motif (His-Ala-Val) that disrupts N-cadherin extracellular interactions (### p<0.0001, significant interaction between peptide treatment and time, two-way RM ANOVA, F(78,624) = 4.037, *p< 0.05, Bonferroni’s test post hoc, n=8 cells/8 mice vehicle, 6 cells/6 mice HAV, 5 cells/5 mice HAV-S). Vehicle-only and scramble peptide (HAV-S) had no effect on LTP. Data shown as mean ± SEM.
Figure 2
Figure 2. Cocaine-induced conditioned place preference (CPP) leads to recruitment of cadherin and GluA1 to excitatory synapses onto dopaminergic neurons in the VTA
(a) Experimental schedule for CPP and extinction experiments. (b) Cocaine administration produced robust CPP (#p<0.001, significant interaction between treatment and test day, two-way RM ANOVA, F(6,60) = 4.422, **p<0.01, Bonferroni’s test post hoc) that extinguished over 5 days. (c) Electron micrograph of VTA synapse showing immunogold-labelled DAT, PSD-95 and cadherin (Scale bar = 100 nm). (d) Cadherin localization shifted to the synaptic membrane of excitatory synapses following cocaine CPP (20 nm bins). (e) The relative percentage of cadherin at the synaptic membrane at excitatory synapses ([#cadherin beads within 40 nm of the pre and postsynaptic membrane]/[# beads within 500 nm of the synaptic membrane], expressed as a percent relative to saline controls) was significantly increased compared to saline controls following cocaine-induced CPP, (p<0.01, significant interaction, two-way ANOVA, F(4,41) = 4.999, **p< 0.01, Bonferroni’s test post hoc), but not in home cage controls, following extinction of CPP, following return to home cage for 6 days after CPP (CPP + HC), or following food CPP (NF: No food, PF: Palatable food, see also Supplementary Fig. 6). CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP+Extinction: n = 5 mice saline, 6 mice cocaine; CPP+HC: n = 6 mice saline, 5 mice cocaine; Food CPP: n = 4 mice NF, 6 mice PF. (f) The percentage of cadherin localized to the synaptic membrane at excitatory synapses was significantly correlated with time spent in conditioned chamber following cocaine CPP (Linear regression, p<0.05, F(1,10) = 7.758), 6 mice per condition. (g) No change in cadherin localization was observed at inhibitory synapses following cocaine CPP. (h) The relative percentage of cadherin at the synaptic membrane was not changed at inhibitory synapses following cocaine CPP, home cage controls, extinction of CPP, or Food CPP. CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP+Extinction: n = 4 mice saline, 5 mice cocaine; CPP+HC: n = 6 mice saline, 6 mice cocaine; Food CPP: n = 4 mice NF, 6 mice PF (i) Cadherin localized to synaptic membrane at inhibitory synapses was not correlated with time spent in conditioned chamber following CPP. (j) GluA1 localization shifted towards the PSD (postsynaptic density) membrane following cocaine CPP. (k) The relative percentage of GluA1 at the PSD membrane ([# GluA1 beads within 30nm of the PSD membrane]/[# beads within 500nm of the PSD membrane], expressed as a percent relative to saline controls) was significantly increased at excitatory synapses following cocaine-induced CPP (p<0.01, significant interaction, two-way ANOVA, F(4,44) = 4.049, **p< 0.01, Bonferroni’s test post hoc), but not in home cage controls, following extinction of CPP, following return to home cage for 6 days after CPP, or following food CPP. CPP: n = 6 mice saline, 6 mice cocaine; HC: n = 4 mice saline, 3 mice cocaine; CPP+Extinction: n = 6 mice saline, 6 mice cocaine; CPP+HC: n = 6 mice saline, 6 mice cocaine; Food CPP: n = 6 mice NF, 5 mice PF (l) Percentage of GluA1 localized to the PSD membrane was significantly correlated with time spent in conditioned chamber following cocaine-induced CPP (Linear regression, p<0.05, F(1,10) = 7.848), n = 6 mice per condition. >100 synapses were analyzed per group. Data shown as mean ± SEM with individual mice (circles) overlaid.
Figure 3
Figure 3. Stabilization of cadherin by β-catenin at synapses in the VTA reduces cocaine-induced CPP
(a) β-catenin levels were significantly increased in DAT+ neurons (arrowheads) in the VTA of DAT-Cre;β-catΔex3 mice compared to adjacent DAT- cells (asterisks) and DAT+ neurons in control mice (p<0.001, significant interaction between genotype and cell type, two-way ANOVA, F(1,48) = 38.13, ***p<0.001 Bonferroni’s test post hoc, WT: n=14 cells non-DAT, 10 cells DAT+, DAT-Cre;β-catΔex3: 16 cells non-DAT, 12 cells DAT+). (b) Cocaine-induced CPP was significantly reduced in DAT-Cre;β-catΔex3 mice compared to controls (#p<0.05, significant interaction between genotype and test day, two-way RM ANOVA, F(6,264) = 2.194, * p<0.05, Bonferroni’s test post hoc, n=23 mice per genotype). Preference for the cocaine-paired chamber returned to baseline after 3 days of extinction in control mice and 1 day of extinction in DAT-Cre;β-catΔex3 mice (+p<0.01, ++p<0.0001, significantly different from day 1, Dunnett’s test post hoc). DAT-Cre;β-catΔex3 mice showed no differences in average speed (c) in the three-chamber CPP apparatus during habituation or after CPP (n=10 mice control, 14 mice DAT-Cre;β-catΔex3), and no differences in locomotor sensitization to cocaine (d) compared to littermate controls (n = 16 mice control, 16 mice DAT-Cre;β-catΔex3). (e) DAT-Cre;β-catΔex3 mice showed normal coordination and motor learning on an accelerating rotarod task (n=13 mice control, 15 mice DAT-Cre;β-catΔex3). (f) DAT-Cre;β-catΔex3 mice showed no change in the acquisition of contextual fear memory following a foot shock in a novel environment compared to littermate controls, indicating intact spatial memory formation (n=16 mice control, 16 mice DAT-Cre;β-catΔex3) (g) DAT-Cre;β-catΔex3 mice showed no impairments in CPP driven by palatable food rewards (n = 17 mice control, 12 mice DAT-Cre;β-catΔex3). Data shown as mean ± SEM,.
Figure 4
Figure 4. Stabilization of cadherin at synapses in the VTA prevents the removal of GluA2-containing AMPARs, and blocks the insertion of GluA1-containing AMPARs
Immunogold EM was used to identify differences in cadherin, GluA2, and GluA1 localization after cocaine CPP in wildtype and DAT-Cre;β-catΔex3. (a, b) Cadherin localization to the synaptic membrane was increased under basal conditions in DAT-Cre;β-catΔex3 mice, and recruitment of additional cadherin to the synaptic membrane during CPP was blocked (p<0.05, significant interaction between treatment and genotype, two-way ANOVA, F(1,8) = 5.613, n=3 mice per condition. >100 synapses were analyzed per group). (c, d) The removal of GluA2 from the PSD membrane at excitatory synapses following CPP was blocked in DAT-Cre;β-catΔex3 mice (p<0.01, significant interaction between treatment and genotype, two-way ANOVA, F(1,8) = 22.07, n=3 mice per condition). (e, f) The insertion of GluA1 to the PSD membrane at excitatory synapses onto dopaminergic neurons following CPP was blocked in DAT-Cre;β-catΔex3 mice (p<0.01, significant interaction between treatment and genotype, two-way ANOVA, F(1,11) = 10.75, n=3 mice control saline, n=3 mice control cocaine; n=5 DAT-Cre;β-catΔex3 mice saline, 4 DAT-Cre;β-catΔex3 mice cocaine). a, c, e: *p< 0.05, **p< 0.01 Bonferroni’s test post hoc. Data shown as mean ± SEM with individual mice (circles) overlaid.
Figure 5
Figure 5. Stabilization of cadherin at synapses in the VTA blocks LTP by retaining GluA2-containing AMPARs and preventing the insertion of GluA2-lacking AMPARs
(a) Spike-timing-dependent LTP in the VTA was abolished in naïve DAT-Cre;β-catΔex3 mice (#p<0.001, significant interaction between genotype and time, two-way RM ANOVA, F(52, 468) = 2.644, * p<0.05, Bonferroni’s test post hoc, n=5 cells/5 mice WT, 6 cells/6 mice DAT-Cre;β-catΔex3). Treatment with NASPM reversed LTP in control mice, but had no effect on EPSP amplitude in DAT-Cre;β-catΔex3 mice. (b) Increased AMPAR/NMDAR ratio 24 h after cocaine administration observed in control mice was abolished in DAT-Cre;β-catΔex3 mice (p<0.05, two-way ANOVA, significant interaction between genotype and drug treatment, F(1,39) = 5.143, *p<0.05, Bonferroni’s test post hoc, n = 10 cells/4 mice control saline, 11 cells/5 mice control cocaine, 12 cells/6 mice DAT-Cre;β-catΔex3 saline, 10 cells/5 mice DAT-Cre;β-catΔex3 cocaine). (c) Increased rectification index of AMPAR EPSCs 24 h after cocaine administration observed in control mice was absent in DAT-Cre;β-catΔex3 mice (p<0.01, two-way ANOVA, significant interaction between genotype and drug treatment, F(1,31) = 7.641, **p<0.01 Bonferroni’s test post hoc, n = 9 cells/3 mice control saline, 8 cells/3 mice control cocaine, 9 cells/3 mice DAT-Cre; β-catΔex3 saline, 9 cells/3 mice DAT-Cre;β-catΔex3 cocaine). The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) (d) and miniature inhibitory postsynaptic currents (mIPSCs) (e) onto dopaminergic neurons in the VTA were unchanged in DAT-Cre;β-catΔex3 mice compared to controls, indicating that basal excitatory and inhibitory synaptic transmission at these synapses was unaltered in the VTA of DAT-Cre;β-catΔex3 mice (n = 9 cells/3 mice control, 8 cells/3 mice DAT-Cre;β-catΔex3, unpaired t-tests, p>0.05). Data shown as mean ± SEM with individual cells (circles) overlaid.
Figure 6
Figure 6. Model of changes in cadherin and AMPAR subunit localization in control and DAT-Cre;β-catΔex3 mice during conditioned place preference (CPP)
(a–b) Wildtype mice. (a) Under basal conditions, the population of AMPARs at excitatory inputs to dopaminergic neurons is composed of GluA1/2 heteromers. Cadherins regulate the dynamic localization of AMPARs through direct and indirect interactions with GluA1 and GluA2 –,. (b) During cocaine-mediated CPP, activity is enhanced at excitatory inputs to dopaminergic neurons, driving the removal of GluA1/2 heteromers and the insertion of Ca2+-permeable GluA1 homomers into AMPA receptor ‘slots’ within the PSD,. Enhanced synaptic activity also leads to increased levels of cadherin at the synaptic membrane. Cadherins are then situated to associate with and stabilize GluA1 homomers at the synaptic membrane, contributing to the potentiation of these synapses underlying behavioral changes in CPP. (c–d) DAT-Cre;β-catΔex3 mice. (c) Under basal conditions, elevated levels of β-catenin promote the stability of cadherin, resulting in a significant increase in cadherin localized to the synaptic membrane. Cadherins associate with GluA1/2 heteromers, enhancing their stability at the synaptic membrane. (d) During cocaine-mediated CPP, the removal of GluA1/2 heteromers is prevented due to their stabilization by increased synaptic cadherin in DAT-Cre;β-catΔex3 mice. GluA1/2 heteromers are retained in available AMPAR ‘slots’, preventing the insertion of GluA1-containing AMPARs and the potentiation of these synapses. Thus, stabilizing synaptic cadherin in DAT-Cre;β-catΔex3 mice disrupts the cocaine-induced switch in AMPAR composition and significantly reduces CPP.

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